The utility of immunoadjuvants (or "adjuvants") in the administration of immunogenic substances has long been recognized, and considerable work has been done to discover substances which, when added to an antigen or other immunogenic substance, would potentiate antigenic activity and thereby antibody stimulating capacity. To date, many such adjuvants have been discovered, such as the use of alum precipitation of antigens; combining certain specific antigens, some of which would potentiate the activity of the others in the mixture; the use of calcium phosphate, particularly to potentiate influenza antibody production, and the similar use of Staphylococcus toxin, which appears to improve the antibody response to certain antigens. Several other adjuvant substances also have been considered such as tapioca, calcium or magnesium salts, tannin, and the like, which when added to certain specific antigens can increase the antibody titer over that obtainable when the antigen alone is administered.
Immunoadjuvants increase the amount of antibody produced and reduce the quantity of antigen necessary for injection and thus, the frequency of injection. Aluminum adjuvants are widely used, and although considered safe in man, sterile abscesses and persistent nodules may follow their use. Complete Freund's adjuvant, an oil-in-water emulsion containing tubercle bacilli, is more potent than the aluminum adjuvants. However, the deleterious side effects, including severe granuloma formation, allergic responses, and oil retention in the tissues, preclude its use in man.
Interleukin-2 (IL-2) occupies a central role in the augmentation of cell mediated immune responses. See K. A. Smith, Science, 240, 1169 (1988). It has recently been demonstrated that when used as a vaccine adjuvant, IL-2 overcomes genetic nonresponsiveness to malaria sporozoite peptides and enhances protection against Herpes simplex and rabies viruses. See, for example, M. F. Good et al., J. Immunol., 141, 972 (1988); A. Weinberg et al., J. Immunol., 140, 294 (1988); J. H. Nunberg et al., J. Cell. Biochem., supp 128, 12 (1988). IL-2 also facilitates nonspecific tumor killing by activated macrophages, and induction of the lymphokine activated killer (LAK) phenomenon in lymphocytes. See, for example, M. Malkovsky et al., Nature, 32.5, 262 (1987); E. A. Grimm et al., J. Exp. Med., 158, 1356 (1988); J. J. Mule et al., Science, 225., 1487 (1984); and J. M. Zarling et al., Nature, 274, 269 (1978). It has exhibited antineoplastic activity in numerous murine tumor models when used alone or in combination with adoptively transferred cells, i.e., cells stimulated with IL-2 that exhibit lymphokine activated killer (LAK) activity. However, when this lymphokine has been utilized alone or in combination with peripheral blood mononuclear cells stimulated with IL-2 in tissue culture media, there has been limited success in human cancer immunotherapy protocols. See R. R. Salup et al., Cancer Immunol. Immunother., 22, 31 (1986); N. Berinstein et al., J. Immunol., 140, 2839 (1988); S. A. Rosenberg et al., N. Engl. J. Med., 313, 1485 (1985); ibid., 316, 889 (1987); Ann. of Surg., 208, 121 (1988), Ann. Int. Med., 108, 853 (1988); and R. I. Fisher et al., Ann. Int. Med., 108, 518 (1988). The clinical responses of patients receiving IL-2 and/or adoptively transferred cells stimulated with IL-2 have been in the range of about 20% to 30% for renal cell carcinoma, 10% to 20% for melanoma, and 15% or less for colon carcinoma.
Severe systemic toxicity has been associated with high dose, prolonged IL-2 administration in man and with protocols utilizing adoptively transferred cells with LAK activity in combination with IL-2. Side effects have included fever, malaise, hepatic and renal dysfunction, central nervous system adverse effects such as somnolence, disorientation, and coma, and anasarca associated with a life-threatening pulmonary capillary leak syndrome. See S. A. Rosenberg, in Important Advances in Oncology, V. T. DeVita et al., eds., J. P. Lippincott Co., Philadelphia, Pa. (1988) at pages 217-257. Furthermore, the half-life of IL-2 in vivo is only about 4 minutes.
The MC-38 murine colon adenocarcinoma, induced by subcutaneous injections of dimethylhydrazine in syngeneic C57BL/6 mice, has been used to evaluate therapeutic efficacy of immunotherapy treatment regimens against hepatic metastases of colon cancer. Significant tumor reduction in this model has been previously achieved with IL-2 activated tumor-infiltrating lymphocytes (TIL), a subpopulation of lymphocytes that infiltrate into growing cancers, in combination with IL-2 and cyclophosphamide. See S. A. Rosenberg et al., Science, 233, 1318 (1986). High doses of IL-2 alone, however, have no significant therapeutic effect on this tumor. Furthermore, a larger number of LAK cells are required to achieve tumor reduction when compared to the number of TIL cells required. Since expansion of both types of cell cultures is difficult, large numbers of cells in starting cultures, or prolonged culture times, are required in order to obtain sufficient numbers of cells for effective immunotherapy.
Liposomes, phospholipid vesicles with either one or more bilayers, profoundly modify and alter the absorption and distribution of entrapped drugs by virtue of lymphatic absorption and macrophage uptake. This uptake occurs mainly in the liver and to a lesser extent in other tissues rich in macrophages including lung, bone marrow, and spleen. See K. J. Hwang, in Liposomes from Biophysics to Therapeutics, M. J. Ostro, ed., Marcel Decker, NY (1987) at pages 109-156. For example, it has been reported that the antifungal efficacy is increased and the systemic toxicity of amphotericin B is significantly reduced in a liposomal formulation. See G. Lopez-Berestein, Ann. Int. Med., 105, 130 (1986). However, the effect of liposomal incorporation and delivery on the localization and activity of cytokines and other bioactive compounds is highly unpredictable. Furthermore, liposomal stability may be adversely affected by interactions with the bioactive compound or its carrier with the phospholipid vesicle wall, leading to low levels of incorporation, leakage of the active ingredient, or low stability of the finished composition. In certain instances there is evidence that cytokine leakage from the liposomes, rather than internalization by macrophages, is associated with biologic activity.
Therefore, a continuing need exists both for immunoadjuvants of increased efficacy and reduced toxicity, as well as to improve the bioactivity and bioavailability of IL-2, while moderating its toxicity. Furthermore, a continuing need exists both for improved varieties of adoptive cells for immunotherapy of cancer and reduced toxicity associated with their use. Additionally, improvements in the drug delivery of cytokines such as IL-2 are needed in order to develop practical outpatient treatment regimens.